Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded-nucleic acid analogs able to modulate gene expression through steric blocking of complementary RNA. PPMO are composed of two components, an antisense morpholino oligomer cargo covalently conjugated to a cell- penetrating delivery peptide. PPMO are completely nuclease-resistant, water-soluble and enter cells without assistance. These characteristics represent distinct advantages over other antisense technologies. PPMO have shown considerable potential against a wide range of clinical targets, including several genetic disorders and numerous viral pathogens. However, the challenge of intracellular endosomal trapping of PPMO is hampering continued clinical development of this class of antisense therapeutics, requiring increased doses of PPMO to achieve clinical efficacy, and narrowing the therapeutic window for this promising technology. The primary objective of this project is to improve the PPMO therapeutic window through the novel combination of an established cell-penetrating peptide with endosome-disruptive viral fusion peptide motifs. We hypothesize that this will enhance intracellular release, thus improving the access of the antisense cargo to its RNA targets within the cytosol of cells. As a model system to validate this innovative delivery approach, we will assess in vitro efficacy using our previously established dengue virus model. Dengue virus is a complex human pathogen currently causing a major global public health problem, yet despite the acute need no licensed antiviral to treat dengue virus infections is currently available. Through this application, we have assembled a multidisciplinary team of researchers with unique expertise in nucleic acid delivery technologies (Dr. Moulton, Oregon State University), as well as an industry partner (Dr. Amanna, Najt Technologies, Inc.), each team with >10 years of experience in early-stage antiviral clinical development. By significantly improving the delivery and activity of the PPMO platform, this project is expected not only to enhance the utility of PPMO as a therapeutic class, but also to provide a novel strategy for addressing dengue virus infections in humans. At the conclusion of this project, we anticipate a novel and improved PPMO platform that can be applied to a wide range of clinical targets.